Exploring the virulence potential of Staphylococcus aureus CC121 and CC152 lineages related to paediatric community-acquired bacteraemia in Manhiça, Mozambique

Staphylococcus aureus is a frequent agent of bacteraemia. This bacterium has a variety of virulence traits that allow the establishment and maintenance of infection. This study explored the virulence profile of S. aureus strains causing paediatric bacteraemia (SAB) in Manhiça district, Mozambique. We analysed 336 S. aureus strains isolated from blood cultures of children younger than 5 years admitted to the Manhiça District Hospital between 2001 and 2019, previously characterized for antibiotic susceptibility and clonality. The strains virulence potential was evaluated by PCR detection of the Panton-Valentine leucocidin (PVL) encoding genes, lukS-PV/lukF-PV, assessment of the capacity for biofilm formation and pathogenicity assays in Galleria mellonella. The overall carriage of PVL-encoding genes was over 40%, although reaching ~ 70 to 100% in the last years (2014 to 2019), potentially linked to the emergence of CC152 lineage. Strong biofilm production was a frequent trait of CC152 strains. Representative CC152 and CC121 strains showed higher virulence potential in the G. mellonella model when compared to reference strains, with variations within and between CCs. Our results highlight the importance of monitoring the emergent CC152-MSSA-PVL+ and other lineages, as they display important virulence traits that may negatively impact the management of SAB paediatric patients in Manhiça district, Mozambique.

Staphylococcus aureus is a frequent agent of bacteraemia.This bacterium has a variety of virulence traits that allow the establishment and maintenance of infection.This study explored the virulence profile of S. aureus strains causing paediatric bacteraemia (SAB) in Manhiça district, Mozambique.We analysed 336 S. aureus strains isolated from blood cultures of children younger than 5 years admitted to the Manhiça District Hospital between 2001 and 2019, previously characterized for antibiotic susceptibility and clonality.The strains virulence potential was evaluated by PCR detection of the Panton-Valentine leucocidin (PVL) encoding genes, lukS-PV/lukF-PV, assessment of the capacity for biofilm formation and pathogenicity assays in Galleria mellonella.The overall carriage of PVL-encoding genes was over 40%, although reaching ~ 70 to 100% in the last years (2014 to 2019), potentially linked to the emergence of CC152 lineage.Strong biofilm production was a frequent trait of CC152 strains.Representative CC152 and CC121 strains showed higher virulence potential in the G. mellonella model when compared to reference strains, with variations within and between CCs.Our results highlight the importance of monitoring the emergent CC152-MSSA-PVL + and other lineages, as they display important virulence traits that may negatively impact the management of SAB paediatric patients in Manhiça district, Mozambique.
Staphylococcus aureus is one of the major causes of bacteraemia and other infections in humans 1 .The versatility of this pathogen is partly determined by a varying repertoire of virulence factors, including toxins that increase the pathogen potential to cause a range of diseases 2 .Clinically important toxins encoded by S. aureus comprise the toxic shock syndrome toxin-1 (TSST-1), staphylococcal enterotoxins (SEs), exfoliative toxins (ETs), and the Panton-Valentine leucocidin (PVL), encoded by the lukS-PV/lukF-PV genes 2,3 .PVL is a bi-component exotoxin that forms pores in the membrane of leukocytes, inducing their lysis 4 .Although carriage of the lukS-PV/lukF-PV genes has been related to specific types of infections, such as skin and soft tissue infections and necrotizing pneumonia, the role of PVL in bacteraemia is still controverse 5,6 .
Biofilm production is another important virulence determinant in S. aureus 7 .Overall, biofilm producing strains are estimated to be associated with 65-80% of all bacterial infections in humans 8 .The biofilm formation is a complex process, in which several components participate, such as the polysaccharide intercellular adhesin (PIA), encoded by the ica operon, and S. aureus surface protein G (encoded by sasG gene) 7 .Those infections are notorious for their chronicity and resilience against therapy, with a consequent increase in morbidity and mortality 7 .Therefore, a better understanding of staphylococcal biofilms is imperative to generate new treatment strategies for biofilm-associated infections, and to reduce their significant impact on disease.
Despite the importance of PVL and biofilm on the S. aureus pathogenesis, most of the studies are based on the epidemiology of the disease, on antimicrobial susceptibility determination, or screening of genes encoding specific resistance determinants and virulence factors, with scarce information regarding the in vitro or in vivo virulence potential of important circulating strains.Several studies evaluated the virulence potential of clinical S. aureus, but only a few were based on infection models that generate in vivo data quickly and inexpensively, such as the Galleria mellonella larval infection model [9][10][11][12][13] .The increasing use of this model is supported by its innate immune response which is similar to the one of mammals, including similar mechanisms of pathogen killing 13,14 .However, most of these reports determining the virulence potential of S. aureus strains are from high and middle-income countries 9,11,12 .As the circulating S. aureus clonal lineages differ from region to region, studies determining the virulence potential of the main clonal lineages circulating in Africa are needed, particularly those associated with severe disease (e.g.bacteraemia).We recently reported the epidemiology and clinical characteristics of children with S. aureus bacteraemia (SAB) over two decades (2001-2019) in the Manhiça district, Mozambique 15 .Afterwards, the SAB-related S. aureus strains (N = 336) were analysed for their clonality, antibiotic resistance phenotypes and resistance determinants 16 .The data gathered throughout the two decades of the study period revealed an evolution in the predominant clonal lineages over time.Particularly, we observed a predominance of methicillin-susceptible S. aureus (MSSA)-clonal complex (CC)152 and MSSA-CC121 in the last six years of the study (2014-2019); in contrast to the predominance of CC1, CC5, CC8, CC15, CC25, CC80 and CC88 between 2001 and 2013.
The present study aimed to evaluate the virulence profile of the CC121 and CC152 S. aureus related to SAB in our setting.The strains virulence profile was characterized by screening the genes encoding for PVL toxin, the biofilm-associated genes icaADB, atlA and sasG, and evaluating the strains capacity to produce biofilms, whereas the virulence potential of representative strains was assessed in the G. mellonella larval infection model.

Frequency of lukS-PV/lukF-PV genes among SAB-related S. aureus strains
Overall, the lukS-PV/lukF-PV genes were detected in 43.7% (147/336) of the S. aureus strains.The comparative analysis of the presence of lukS-PV/lukF-PV genes and the MLST CCs previously described for this collection 16 revealed predominance of these genes among strains belonging to CC22 (100%), CC152 (96.9%) and CC121 (85.3%), followed by CC88 (69.2%),CC80 (62.5%) and CC1 (47.2%) (Table 1).The frequency of lukS-PV/lukF-PV genes among the remaining CCs varied between 0 and 40%.Analysing the variation in the frequency of detection of lukS-PV/lukF-PV genes throughout the study period, we could observe two distinct trends (Fig. 1).During the first twelve years of the study (2001-2013), the frequency of PVL-positive strains varied between ~ 20 and ~ 60%.In the following years (2014-2019), we observed an increasing trend in PVL-positive strains, accounting from ~ 70% up to 100% of all SAB-related S. aureus strains isolated in that timeframe.Of particular interest, the increasing trend of PVL was linked to the emergence of the CC152 strains.
The screening of biofilm-associated genes showed that icaADB, part of the ica operon, were present in all CC121 and CC152 strains tested, including non-and strong-biofilm producers.The atlA gene, encoding a major staphylococcal autolysin was present in 98.5% (64/65) of strains tested.The single strain that did not carry this  www.nature.com/scientificreports/gene was a CC121 biofilm strong producer.The sasG gene was absent from most strains, with an overall frequency of 7.7% (5/65), corresponding to two CC121 (5.9%, 2/34) and three CC152 (9.1%, 3/33) strains.Overall, these results indicate that the presence or absence of these genes in CC121 and CC152 is not directly related to the biofilm producing phenotype observed.

Virulence potential of representative CC121 and CC152 S. aureus strains in the G. mellonella infection model
The virulence potential of eleven representative strains of the emergent lineages circulating in our setting, CC121 and CC152, was assessed in the larval G. mellonella infection model.Overall, all clinical strains tested showed high virulence in G. mellonella when compared to the reference strains S. aureus ATCC ® 25923™ and S. aureus RN4220.Analysing the survival curves and median survival time, we observed a variation of the virulence potential within and between the CCs analysed.Comparing CC121 strains, SA 199 (PVL + ) was the most virulent, followed by SA 152 (PVL + ) > SA 179 (PVL + ) > SA 352 (PVL − ) > SA 243 (PVL + ) (Fig. 3).Similarly, CC152 strains could be differentiated regarding their virulence potential as follows, SA 301 (PVL + ) > SA 302 (PVL − ) > SA 330 (PVL + ) > SA 343(PVL + ) > SA 353 (PVL + ) > SA 324 (PVL + ).For all strains, the median survival time was inversely proportional to the bacterial inoculum; from 2 to > 7 days at 10 5 CFU/larva to only 1 day at 10 7 CFU/larva (Table 2).In agreement with our findings, previous reports described mortality of G. mellonella larvae infected with S. aureus to be dose-dependent, in which infection with 1 × 10 7 CFU/larva resulted in death after 24 h, whereas infection with 1 × 10 5 CFU resulted in ∼20% of deaths after 120h 17 .
When analysing the overall virulence potential of S. aureus CC121 and CC152 lineages, no relation was found with the virulence traits studied (carriage of lukS-PV/lukF-PV, biofilm-associated genes or biofilm production phenotype) or with the patient outcome, i.e. higher virulence potential did not correlate with poorer patient outcome, and vice-versa (Table 2).
We also analysed the virulence potential of an additional set of four strains, representative of CC8 clonal lineage.Although present in lower frequency, this lineage comprises the majority (15 out of 16) of the MRSA strains detected in our setting over the two decades of the study period (Table 1).Similarly to the other CCs analysed, the CC8 strains (ST8 and ST612) showed a virulence potential higher than the reference strains.Within this clonal complex, the ST8 strains (SA 215, MSSA; SA 277, MRSA) were the most virulent, compared to the two ST612-MRSA strains, SA 276 and SA 188 (Supplementary Fig. S1 and Supplementary Table S1).In general, the CC8 strains showed similar virulence potential to the CC121 and CC152 strains.

Discussion
We recently reported the high diversity of S. aureus clonal lineages from children with bacteraemia admitted to the Manhiça District Hospital (MDH), Mozambique with predominance of the CC121 and CC152 (emergent clone) in the last years of the study period, using the surveillance system established in this setting 16 .These same clones were recently detected circulating in other African countries 18 , suggesting their important role in the epidemiology of staphylococcal invasive infections in this region.This is one of the few studies assessing the virulence potential of SAB-related S. aureus in African countries.Despite the absent correlation between the bacterial virulence traits analysed and the median survival time of G. mellonella, a higher virulence potential was established for the SAB-related S. aureus strains circulating in Manhiça District, compared to reference strains, particularly for strains from the emergent CC152 lineage.The same pattern of higher virulence potential was detected for other CC8 clinical strains from our collection (Supplementary Fig. S1 and Table S1).Several virulence factors carried in S. aureus are often encoded on the pathogen's accessory genome.The clinically most important toxins encoded by S. aureus are SEs, TSST-1, ETs and PVL, responsible for necrotic lesions involving skin and mucosa, and necrotic haemorrhagic pneumonia 2,3 .Almost half (43.7%) of the entire S. aureus study collection carried the PVL determinants lukS-PV/lukF-PV and these predominated among specific clonal lineages.In our study, these genes predominated among CC121, CC152 and CC22 lineages, supporting the observation of previous reports [18][19][20][21] .The increase of PVL in the last years of the surveillance period was most likely due to the increase of the CC152-MSSA-PVL + , although some caution should be taken in this analysis, considering the low rates of S. aureus isolated in this period.The absence of a relation between biofilm production, presence of biofilm-associated genes (icaADB, atlA, sasG), PVL encoding genes (lukS-PV/lukF-PV), resistance profile and the virulence of either CC121 or CC152 strains in G. mellonella infection model, suggests the contribution of additional virulence factors to the overall virulence potential.Noteworthy, in our study, lukS-PV/lukF-PV genes predominated from both MDR and non-MDR strains and were absent among MRSA strains.We have previously found that infection by these MDR strains was associated to higher mortality of SAB patients in our setting 15 .In the present study, infection of G. mellonella by MDR strains did not result in a significantly different survival time when compared with infection by non-MDR strains.
Our results revealed a high virulence potential of the CC152 lineage predominantly circulating in Manhiça in the last years of the study 16 .This finding is matter of concern since CC152 has been reported as an important emergent and prevalent clone in several African countries 18,22 .CC121, for which we have also established a high virulence potential, is a clonal lineage also increasingly identified in the African continent 18,23 .Further detailed epidemiological and molecular analysis of these important clones is urgently needed for prompt adoption of adequate prevention and control measures.

Conclusions
To our knowledge, this is one of the first studies documenting the virulence potential of SAB-related S. aureus from Mozambique.Here we report a high frequency of MSSA but not MRSA strains carrying the determinants for PVL toxin.Strains from the lineage CC152 and, in a lesser extent, CC121, that have established in our setting  .c Fully susceptible to the tested antibiotics (Garrine et al. 2023b) 16 .ST sequence type, MDR multidrug-resistant S. aureus (defined as those strains not susceptible to three or more unrelated classes of antibiotic).Significant differences are highlighted at bold type.nd not determined.www.nature.com/scientificreports/ in recent years are frequent strong biofilm producers and show higher virulence potential in the G. mellonella infection model when compared to two prototype reference strains.Although no correlation could be established between particular clonal lineages and virulence, the approach described in this study has potential to be applied to large datasets in future studies.

Patients
Our results highlight the importance of monitoring the emergent CC152-MSSA-PVL + and other lineages as they display important virulence traits that may impact negatively the management of SAB paediatric patients in Manhiça, Mozambique.

Methodology Study design and S. aureus collection
In this study we analysed S. aureus strains isolated from blood cultures of children admitted to the Manhiça District Hospital (MDH, Manhiça District, southern Mozambique) between 2001 and 2019.The pertinent characteristics of our S. aureus collection (N = 336), previously described 15,16 , are presented in Table 1.These strains are part of the 24h morbidity surveillance ongoing in MDH as one of the main activities of the "Centro de Investigação em Saúde de Manhiça" (CISM).The surveillance performs a standardized collection of clinical data among all paediatric patients (< 15 years of age) and a specific microbiological surveillance based on the systematic collection of blood cultures among admitted patients, according to the relevant guidelines and regulations 15,24 .

Evaluation of biofilm production by CC121 and CC152 strains
We evaluated the biofilm mass production for most strains from CC121 (97.1%, 33/34) and CC152 (97.0%, 32/33).The strains capacity for biofilm production was assessed by the crystal violet adhesion method 27 , using the conditions described elsewhere 12 .Briefly, the isolates were cultured in TSB medium and incubated overnight without shaking at 37 °C.A cellular suspension adjusted to ~ 5 × 10 8 CFU/mL in TSB was prepared, diluted 1:100 in TSB supplemented with 1% glucose and 3% NaCl and 0.2 mL aliquots distributed in PS flat-bottom TC 96 well microtiter plates (Orange Scientific, Braine-L' Alleud, Belgium), followed by incubation at 37 °C during 24 h.Afterward, the wells content was discarded and washed with PBS.Adherent bacteria were fixed with methanol (99%) for 20 min and air-dried overnight.The biofilm mass was dyed with crystal violet (0.1%) for 15 min, washed with distilled water and air-dried.The crystal violet was solubilized in acetic acid (33%) for 30 min and the optical density at 570 nm (OD570) was measured in a Synergy HT microplate reader (Biotek, Winooski, VT, USA).Each assay included in quadruplicates, the control strains S. aureus ATCC ® 25923™ (moderate to strong biofilm producer), S. epidermidis ATCC ® 12228™ (weak to moderate biofilm producer, ica -), and S. epidermidis ATCC ® 35984™ (strong biofilm producer, ica + ), as well as the S. aureus strains under testing, a negative control (supplemented TSB), and a blank (33% acetic acid).Biofilm production was also assessed for reference strain S. aureus RN4220 (moderate to strong producer).Biofilm production was categorized according to Stepanović's criteria 27,28 , which establishes a cut-off value (ODc) defined as the geometric mean of OD570 of the negative control (supplemented TSB) + 3 × the corresponding standard deviation (SD).Strains were characterized as follows: OD strain < ODc, biofilm non-producers; ODc < OD strain < 2 × ODc, weak producers; 2 × ODc < OD strain < 4 × ODc, moderate producers; OD strain > 4 × ODc, strong producers.Each assay was performed, at least, in duplicate.To minimize intra-and inter-assay variability, an assay was only validated when associated with an SD value < 20% of the corresponding geometric mean (either considering an individual assay or duplicates) and assigning the strain to the same category.A strain was considered a biofilm producer if assigned to the weak, moderate or strong producer phenotypes.

Assessment of the virulence potential of S. aureus in the G. mellonella infection model
The virulence potential was evaluated for eleven representative strains of CC121 and CC152, selected according to the presence/absence of PVL encoding genes, potential of biofilm production, resistance profile (MDR/non-MDR) and outcome of infected patients (Table 2).The G. mellonella were reared at GHTM/IHMT-NOVA at 28 °C (± 1 °C) under dark with a high nutrient diet (corn flour, dried yeast, soy flour, dry milk, honey, glycerol, bee wax blocks).Larvae at the final instar stage, weighting 300 mg ± 50 mg and with no signs of melanisation were used in the infection assays.We determined the virulence potential of each strain using two distinct bacterial inoculums, 10 5 and 10 7 CFU/larva.The procedures for bacterial inoculum preparation and larvae infection were conducted as described elsewhere 12 .Briefly, bacteria were inoculated in TSB and incubated overnight at 37 °C with agitation.The culture was washed in PBS, resuspended and adjusted to ~ 5 × 10 8 CFU/mL, then diluted in 1:100 PBS to achieve ~ 5 × 10 6 CFU/mL and ~ 5 × 10 4 CFU/mL, respectively.Bacteria enumeration was determined by CFU counting.Each assay included four groups of G. mellonella larvae (n = 10 larvae/group), as follows: (i) non-manipulated; (ii) inoculated with PBS (control); (iii) inoculated with 10 5 CFU/larva; (iv) inoculated with 10 7 CFU/larva.All larvae were inoculated with a 0.02 mL inoculum by injection with an insulin syringe (Omnican 100, Braun) in the last proleg.The G. mellonella survival was monitored each 24 h post-infection for seven days, as previously described 14 .The virulence potential of each staphylococcal strain was evaluated in, at least, three independent infection assays 12 .

Data analysis
Data from virulence assays in the G. mellonella infection model were analysed by Kaplan-Meier survival curves using GraphPad Prism v8.0.1 (San Diego, CA, USA).Survival rates between groups were compared with the Log-Rank (Mantel-Cox) test.Data on S. aureus antibiotic resistance phenotype (MDR/non-MDR; MRSA/MSSA) and clonal lineages 16 as well as the patients outcome (hospital discharge) 15 were compared with virulence traits (lukS-PV + /lukF-PV + , carriage of icaADB, atlA or sasG genes and biofilm production phenotype) using χ 2 or Fisher's exact test as appropriate (STATA version 14.2, LP, College Station, Texas, USA).Wilcoxon rank sum tests were used for nonparametric comparisons (e.g.patients Length of stay, LOS).We deemed a p-value of 0.05 or lower to be statistically significant.

Ethical approval
The S. aureus isolates analysed in this study fall within the scope of the ongoing morbidity and microbiological surveillance system established as part of the Health and Demographic Surveillance System at CISM, approved by the Institutional Ethics Review Board for Health at CISM and the National Bioethics Committee for Health.All residents of Manhiça district have signed individual informed consent forms to participate in the ongoing surveillance.

Figure 2 .
Figure 2. Distribution of CC152 and CC121 strains according to biofilm production phenotype (A); variation and median OD570 nm values (B).

Table 2 .
Main virulence and antibiotic resistance traits of representative SAB-related S. aureus strains selected for the assays in the G. mellonella infection model.a